Department of Biotechnology, School of Agriculture, Tarbiat Modares University, Tehran, Iran.
Prep Biochem Biotechnol. 2011;41(2):175-86. doi: 10.1080/10826068.2011.547371.
Recombinant tissue-type plasminogen activator (rt-PA) has been produced in different hosts. In this research, transgenic tobacco was selected for production of human rt-PA. Transgenic plants were analyzed by polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. The protein was extracted by Lysine Sepharose chromatography column and was further purified by HiTrap desalting column. The function of eluted protein was analyzed on zymography gel. The results showed that the 1.7-kb cDNA of tissue-type plasminogen activator (t-PA) (as well as a shortened 650-bp transcript of t-PA) has been expressed in transgenic plants. The anticipated 63-kD protein band and an additional 53-kD protein were observed in transgenic plants. Finally, zymography assay revealed that the purified rt-PA has anticipated appropriate activity comparable to a positive control drug (Alteplase). On the whole, we can say that transgenic tobacco is a good alternative host for production of t-PA.
重组组织型纤溶酶原激活剂（rt-PA）已在不同的宿主中生产。在这项研究中，选择转基因烟草来生产人源 rt-PA。通过聚合酶链反应（PCR）和逆转录（RT）-PCR 对转基因植物进行分析。通过赖氨酰琼脂糖凝胶柱提取蛋白质，并通过 HiTrap 脱盐柱进一步纯化。通过酶谱凝胶分析洗脱蛋白的功能。结果表明，组织型纤溶酶原激活剂（t-PA）的 1.7-kb cDNA（以及 t-PA 的缩短 650-bp 转录本）已在转基因植物中表达。在转基因植物中观察到预期的 63-kD 蛋白带和另外的 53-kD 蛋白。最后，酶谱分析表明，纯化的 rt-PA 具有预期的适当活性，可与阳性对照药物（Alteplase）相媲美。总的来说，我们可以说转基因烟草是生产 t-PA 的良好替代宿主。
